15 research outputs found
Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen
<p>Abstract</p> <p>Background</p> <p>Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei.</p> <p>Findings</p> <p>Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, <it>Schizosaccharomyces pombe</it>. To preserve <it>in vivo </it>molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates.</p> <p>Conclusions</p> <p>We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.</p
SpyAvidin Hubs Enable Precise and Ultrastable Orthogonal Nanoassembly
The capture of biotin
by streptavidin is an inspiration for supramolecular
chemistry and a central tool for biological chemistry and nanotechnology,
because of the rapid and exceptionally stable interaction. However,
there is no robust orthogonal interaction to this hub, limiting the
size and complexity of molecular assemblies that can be created. Here
we combined traptavidin (a streptavidin variant maximizing biotin
binding strength) with an orthogonal irreversible interaction. SpyTag
is a peptide engineered to form a spontaneous isopeptide bond to its
protein partner SpyCatcher. SpyTag or SpyCatcher was successfully
fused to the C-terminus of Dead streptavidin subunits. We were able
to generate chimeric tetramers with <i>n</i> (0 ≤ <i>n</i> ≤ 4) biotin binding sites and 4-n SpyTag or SpyCatcher
binding sites. Chimeric SpyAvidin tetramers bound precise numbers
of ligands fused to biotin or SpyTag/SpyCatcher. Mixing chimeric tetramers
enabled assembly of SpyAvidin octamers (8 subunits) or eicosamers
(20 subunits). We validated assemblies using electrophoresis and native
mass spectrometry. Eicosameric SpyAvidin was used to cluster trimeric
major histocompatibility complex (MHC) class I:β<sub>2</sub>-microglobulin:peptide complexes, generating an assembly with up
to 56 components. MHC eicosamers surpassed the conventional MHC tetramers
in acting as a powerful stimulus to T cell signaling. Combining ultrastable
noncovalent with irreversible covalent interaction, SpyAvidins enable
a simple route to create robust nanoarchitectures
Bicc1 is a genetic determinant of osteoblastogenesis and bone mineral density
Patient bone mineral density (BMD) predicts the likelihood of osteoporotic fracture. While substantial progress has been made toward elucidating the genetic determinants of BMD, our understanding of the factors involved remains incomplete. Here, using a systems genetics approach in the mouse, we predicted that bicaudal C homolog 1 (Bicc1), which encodes an RNA-binding protein, is responsible for a BMD quantitative trait locus (QTL) located on murine chromosome 10. Consistent with this prediction, mice heterozygous for a null allele of Bicc1 had low BMD. We used a coexpression network–based approach to determine how Bicc1 influences BMD. Based on this analysis, we inferred that Bicc1 was involved in osteoblast differentiation and that polycystic kidney disease 2 (Pkd2) was a downstream target of Bicc1. Knock down of Bicc1 and Pkd2 impaired osteoblastogenesis, and Bicc1 deficiency–dependent osteoblast defects were rescued by Pkd2 overexpression. Last, in 2 human BMD genome-wide association (GWAS) meta-analyses, we identified SNPs in BICC1 and PKD2 that were associated with BMD. These results, in both mice and humans, identify Bicc1 as a genetic determinant of osteoblastogenesis and BMD and suggest that it does so by regulating Pkd2 transcript levels